We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus prevaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4+CD25+CD127-FoxP3+CTLA4+) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS greater than HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted. We have compared the effects of recombinant Saccharomyces cerevisiae (yeast)-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4+CD25+CD127-FoxP3+ regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4+ T cells. CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4+CD25+ activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-alpha, IFN-gamma) and IL-6. In addition, yeast-treated DCs used as APCs incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4+ T cells compared to the use of CD40L-matured DCs used as antigen-presenting cells. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines. Clinical studies are now ongoing with recombinant yeast-CEA vaccine. A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4posCD25highCD127neg Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.